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Isolation of specific tryptic peptides using chip-based bioaffinity reactor

Rok: 2005

Druh publikace: ostatní - přednáška nebo poster

Název zdroje: 2nd IPSO Congress Proteomics and Genomics

Název nakladatele: Italian Proteome Society

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Jazyk	Název	Abstrakt	Klíčová slova
cze	Izolace specifických tryptických peptidů.	Izolace specifických tryptických peptidů.	micro-chip, anhydrotrypsin, izolace, tryptické peptidy
eng	Isolation of specific tryptic peptides using chip-based bioaffinity reactor	Anhydrotrypsin is bioaffinity ligand for highly specific trapping of peptides which correspond to the products generated by the action of various trypsin-like proteases and binds, in slightly acidic conditions, only specific peptides containing arginine and lysine residues at their C-termini 1,2. Anhydrotrypsin is catalytically inactive derivative of bovine trypsin in which serine residue (Ser-195) is first modified with phenylmethanesulfonyl fluoride (PMSF), an active site-directed reagent, and then the modified protein is treated with alkali results in elimination of the modified group and the serine is converted to a dehydroalanine 3. Biospecific carrier prepared by immobilization of anhydrotrypsin on magnetic or non-magnetic micro- and nanoparticles could be used for purification, isolation and preconcentration from complex mixture of tryptic peptides in one step. One of the modern trends in biochemistry is focused on the application of functional spherical nanoparticles for isolation or enzyme modification of target protein directly in a channel of microfluidic device. IMER with immobilized trypsin in tandem with chip-based IMAR with immobilized anhydrotrypsin was used for simplification of peptide mapping including the digestion of protein coupling with purification and preconcentration of peptides for following analysis by HPLC or MS. High purity and preconcentration of peptides isolated on immobilized anhydrotrypsin significantly facilitated the identification of protein fingerprint even for protein in submicromolar concentration.	micro-chip, anhydrotrypsin, isolation, tryptic peptides

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